A number of short peptides with significant activity as inhibitors of cell growth have been isolated from the Indian Ocean sea hare Dolabella auricularia (Bai et al., Biochem. Pharmacology, 40: 1859-1864 (1990); Beckwith et al., J. Natl. Cancer Inst., 85: 483-488 (1993) and references cited therein). These include Dolastatins 1-10 (U.S. Pat. No. 4,816,444, issued to Pettit et al.) and Dolastatin-15 (European Patent Application No. 398558). Dolastatin 15, for example, markedly inhibits the growth of the National Cancer Institute""s P388 lymphocytic leukemia (PS system) cell line, a strong predictor of efficacy against various types of human malignancies.
The exceedingly small amounts of the various Dolastatin peptides present in Dolabella auricularia (about 1 mg each per 100 kg sea hare) and the consequent difficulties in purifying amounts sufficient for evaluation and use, have motivated efforts toward the synthesis of these compounds (Roux et al., tetrahedron 50: 5345-5360 (1994); Shioiri et al., tetrahedron 49: 1913-24 (1993); Patino et al., tetrahedron 48: 4115-4122 (1992) and references cited therein). Synthetic Dolastatin 15, however, suffers from drawbacks which include poor solubility in aqueous systems and the need for expensive starting materials for its synthesis. These, in turn, have led to the synthesis and evaluation of structurally modified Dolastatin 15 derivatives [cf.: Biorg. Med. Chem. Lett. 4: 1947-50 (1994); WO 93 03054; JP-A-06234790; WO 93 23424].
However, there is a need for synthetic compounds with the biological activity of Dolastatin 15 which have useful aqueous solubility and can be produced efficiently and economically.
Compounds of the present invention include cell growth inhibitors which are peptides of Formula I,
A-B-D-E-F-(G)r-(K)s,-L xe2x80x83xe2x80x83(I),
and acid salts thereof, wherein A, B, D, E, F, G and K are xcex1-amino acid residues, and s and r are each, independently, 0 or 1 L is a monovalent radical, such as, for example, an amino group, an N-substituted amino group, a xcex2-Phydroxylamino group, a hydrazido group, an alkoxy group, a thioalkoxy group, an aminoxy group, or an oximato group.
Another aspect of the present invention includes pharmaceutical compositions comprising a compound of Formula I and a pharmaceutically acceptable carrier.
An additional embodiment of the present invention is a method for treating cancer in a mammal, such as a human, comprising administering to the mammal an effective amount of a compound of Formula I in a pharmaceutically acceptable composition.
The present invention relates to peptides having antineoplastic activity. It also includes pharmaceutical compositions comprising these compounds and methods for treating cancer in a mammal, including a human, by administration of these compositions to the mammal.
Dolastatin 15, a peptide isolated from the sea hare Dolabella auricularia, is a potent inhibitor of cell growth. This compound, however, is present in trace quantities in the sea hare, and is thus difficult to isolate. Dolastatin 15 is also expensive to synthesize and suffers from poor aqueous solubility. As shown herein, however, Dolastatin 15 can serve as a starting point for the development of compounds which overcome these disadvantages while retaining antineoplastic activity or exhibiting greater antineoplastic activity than the natural product. Applicants have discovered that certain structural modifications of Dolastatin 15 provide compounds with a surprisingly improved therapeutic potential for the treatment of neoplastic diseases as compared to Dolastatin 10 and Dolastatin 15. Furthermore, the compounds of the present invention can be conveniently synthesized, as described below in detail.
For the purposes of the present invention, the term xe2x80x9cmonovalent radicalxe2x80x9d is intended to mean an electrically neutral molecular fragment capable of forming one covalent bond with a second neutral molecular fragment. Monovalent radicals include the hydrogen atom, alkyl groups, such as methyl, ethyl and propyl groups, halogen atoms, such as fluorine, chlorine and bromine atoms, aryl groups, such as phenyl and naphthyl groups, and alkoxy groups, such as methoxy and ethoxy groups. Two monovalent radicals on adjacent sigma-bonded atoms can also together form a pi bond between the adjacent atoms. Two monovalent radicals may also be linked together, for example, by a polymethylene unit, to form a cyclic structure. For example, the unit-N(R)Rxe2x80x2, wherein R and Rxe2x80x2 are each a monovalent radical, can, together with the nitrogen atom, form a heterocyclic ring. In addition, two monovalent radicals bonded to the same atom can together form a divalent radical, such as an oxygen atom or an alkylidene group, for example, a propylidene group.
For the purposes of the present invention, the term xe2x80x9cnormal alkylxe2x80x9d refers to an unbranched, or straight chain, alkyl group, for example, normal propyl (n-propyl,xe2x80x94CH2CH2CH3).
The compounds of the present invention can be represented by Formula I,
A-B-D-E-F-(G)r-(K)s-L xe2x80x83xe2x80x83(I),
wherein A, B, D, E, F, G, and K are xcex1-amino acid residues; s and r are each, independently, 0 or 1; and L is a monovalent radical such as an amino group, an N-substituted amino group, a xcex2-Phydroxylamino group, a hydrazido group, an alkoxy group, a thioalkoxy group, an aminoxy group, or an oximato group.
The peptides of Formula I are generally composed of L-amino acids but they can contain one or more D-amino acids. In the following discussion, reference to a particular amino acid includes both enantiomers unless a specific enantiomer is indicated. The present compounds can also be present as salts with physiologically-compatible acids, including hydrochloric acid, citric acid, tartaric acid, lactic acid, phosphoric acid, methanesulfonic acid, acetic acid, formic acid, maleic acid, fumaric acid, malic acid, succinic acid, malonic acid, sulfuric acid, L-glutamic acid, L-aspartic acid, pyruvic acid, mucic acid, benzoic acid, glucuronic acid, oxalic acid, ascorbic acid and acetylglycine.
The following is a description of the present invention, including a detailed description of individual components and of methods of using the claimed compounds.
Identity of A
In one embodiment, A is a proline derivative of Formula IIa, 
where na is an integer, preferably 0, 1, 2, or 3. Ra is a monovalent radical, such as a hydrogen atom or an unsubstituted or fluorine-substituted alkyl group, for example a normal, branched or cyclic C1-C3-alkyl group which is, optionally, substituted by from 1 to about 3 fluorine atoms; suitable examples include methyl, ethyl, isopropyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, 1-methyl-2-fluoroethyl, 1-fluoromethyl-2-fluoroethyl or cyclopropyl; methyl, ethyl or isopropyl are preferred;
In this embodiment, R1a is a monovalent radical, such as a hydrogen atom, an alkyl group, such as a methyl, ethyl or propyl group, or a phenyl group. The phenyl group can be substituted; suitable substituents include one or more halogen atoms, with fluorine, chlorine and bromine atoms preferred, C1-C4-alkyl groups, methoxy, ethoxy, trifluoromethyl or nitro groups. Ra and R1a together can also form a propylene bridge. R2a, R3a, R4a and R5a are each, independently, a monovalent radical, such as a hydrogen atom or an alkyl, preferably, methyl, group.
In another embodiment, A is a substituted glycine derivative of Formula IIIa, 
where Ra has the meaning stated for Ra in Formula IIa and, R1a is a monovalent radical, for example, a hydrogen atom or a C1-C6-alkyl group, preferably a methyl, ethyl or propyl group.
In this embodiment, R6a is a monovalent radical, such as an alkyl, substituted alkyl, alkenyl, phenyl or substituted phenyl group. Suitable examples include methoxymethyl, 1-methoxyethyl, 1,1-dimethyl-hydroxymethyl, 1-trifluoromethylethyl, 1-trifluoromethyl-2,2,2-trifluoroethyl, vinyl, and 1-methylvinyl. Phenyl substituents can include one or more halogen atoms, preferably fluorine, chlorine or bromine atoms, and alkyl, methoxy, ethoxy, trifluoromethyl, and nitro groups.
When R1a is an alkyl group, R6a can also be a C1-C6-alkyl, cycloalkyl, unsubstituted benzyl or substituted benzyl group. Suitable benzyl substituents include one or more halogen atoms, such as fluorine, chlorine or bromine atoms, C1-C4-alkyl groups, and methoxy, ethoxy, trifluoromethyl and nitro groups.
R7a is a monovalent radical, preferably a methyl, ethyl or isopropyl group.
In another embodiment, A is an xcex1-amino acid derivative of Formula IVa, 
where ma is an integer, preferably 1 or 2, and Ra and R7a have the meanings stated for these substituents in Formula IIIa.
In another embodiment, A is an xcex1-amino acid derivative of Formula Va, 
where Ra and R7a have the meanings stated for Ra and R7a in Formula IIIa.
In a further embodiment, A is a substituted proline derivative of Formula VIa, 
where Ra and R1a have the meanings stated for Ra and R1a in Formula IIa, and Xa is a monovalent radical, preferably a hydroxyl, alkoxy, for example, methoxy or ethoxy, group or a fluorine atom.
In another embodiment, A is a thiaprolyl derivative of Formula VIIa, 
where Ra, R1a, R2a, R3a, R4a and R5a have the meanings stated for the respective substituents in Formula IIa.
In another embodiment, A is a 1,3-dihydroisoindole derivative of Formula VIIIa
where Ra has the meaning stated for Ra for Formula IIa.
In another embodiment, A is a 2-azabicyclo[2.2. I ]heptane-3-carboxylic acid derivative of Formula IXa, 
where Za is a single or double bond and Ra has the meaning stated for Formula IIa. The 3-carbonyl substituent can have either the exo or endo orientation.
In another embodiment, A is an xcex1-amino acid derivative of Formula Xa, 
where na has the meaning as stated for na for Formula IIa, and R7a and Ra have the meanings as stated for R7a and Ra for Formula IIIa.
Identity of B
B is a valyl, isoleucyl, allo-isoleucyl, norvalyl, 2-tert-butylglycyl or 2-ethylglycyl residue. B can also be an xcex1-amino acid residue of Formula IIb, 
in which R1b and R2b are each a monovalent radical. R1b is, preferably, a hydrogen atom and R2b is, for example, an alkyl, alkoxyalkyl or alkenyl group. In preferred embodiments, R2b is a cyclopropyl group, a normal or branched butyl, preferably tertiary-butyl, group, a methoxymethyl group, a 1-methoxyethyl group or a 1-methylvinyl group. Additionally, R1b and R2b together can be an isopropylidene group.
Identity of D
D is an N-alkylvalyl, N-alkyl-2-ethylglycyl, N-alkyl-2-tert-butylglycyl, N-alkyl-norleucyl, N-alkyl-isoleucyl, N-alkyl-allo-isoleucyl or N-alkyl-norvalyl residue, where the N-alkyl group is preferably a methyl group or an ethyl group.
In another embodiment, D is an xcex1-amino acid residue of Formula IId, 
where Rd has the meaning stated for Ra in Formula IIIa, R1d is a monovalent radical, preferably a hydrogen atom, and R2d is a monovalent radical, for example, an alkyl, alkoxyalkyl or alkenyl group. In preferred embodiments, R2d is a cyclopropyl group, a normal or branched butyl, preferably tertiary-butyl, group, a methoxymethyl group, a 1-methoxyethyl group or a 1-methylvinyl group. such as a cyclopropyl group, a methoxymethyl group, a 1-methoxyethyl group or a 1-methylvinyl group. Additionally, R1d and R2d together can form an isopropylidene group.
Alternatively, D can be a proline derivative of Formula IIId, 
where nd is an integer, for example, 1 or 2, and R3d has the meaning stated for R1a in Formula IIIa. Xd is a monovalent radical, preferably a hydrogen atom, and, in the case where nd equals 1, can also be a hydroxy or alkoxy, for example, methoxy or ethoxy, group or a fluorine atom.
Identity of E
E is a prolyl, thiazolidinyl-4-carbonyl, homoprolyl or hydroxyprolyl residue, or a cyclic xcex1-amino carboxylic acid residue of Formula II, 
where ne is an integer, preferably 0, 1 or 2. R1 has the meaning stated for R1a in Formula IIIa. R2e and R3e are each a monovalent radical, and can be, independently, a hydrogen atom or an alkyl, preferably methyl, group. R4e is a monovalent radical, preferably a hydrogen atom, a hydroxy, alkoxy, for example, methoxy or ethoxy, group or a fluorine atom. R5e is a monovalent radical, preferably a hydrogen atom or a fluorine atom. In the case where n, is 1, R3e and R4e can together form a double bond, or R4e and R5e can together be a double-bonded oxygen radical. In the case where ne has the value 1 or 2, R1e, and R2e can together form a double bond.
In another embodiment, E is a 2-or 3-amino-cyclopentanecarboxylic acid residue of Formula IIIe, 
where Re is an alkyl group, such as methyl or ethyl, and R1e has the meaning stated for R1a in Formula IIa.
Identity of F
F is a prolyl, thiazolidinyl-4-carbonyl, homoprolyl or hydroxyprolyl residue. F can also be a cyclic xcex1-amino acid residue of Formula IIf, 
where nf is an integer, preferably 0, 1 or 2. R1f f has the meaning stated for R1a in Formula IlIa. R2f and R3f are each a monovalent radical, and can be, independently, a hydrogen atom or an alkyl, preferably methyl, group. R4f is a monovalent radical, preferably a hydrogen atom, a hydroxy, alkoxy, for example, methoxy or ethoxy, group or a fluorine atom. R5f is a monovalent radical, preferably a hydrogen atom or a fluorine atom. In the case where nf has the value 1, R3f and R4f together can form a double bond or R4f and R5f can together be a double-bonded oxygen radical. In the case where nf has the value 1 or 2, R1f and R2f can together form a double bond.
In another embodiment, F is a 2-or 3-amino-cyclopentanecarboxylic acid residue of Formula IIIf
where Rf is a monovalent radical, such as a methyl or ethyl group, and R1f has the meaning stated for R1f in Formula IIIa.
In another embodiment, F is an N-alkylglycyl or N-alkylalanyl residue, and the alkyl group is, preferably, a methyl group or an ethyl group.
Identity of G
G is an xcex1-amino acid residue of Formula IIg, 
wherein R1g is a hydrogen atom, or an alkyl group, for example, methyl, ethyl or n-propyl. R2g can be, for example, a hydrogen atom, or an alkyl, arylalkyl, heteroarylalkyl or aryl group. Preferably, R2g is an ethyl, isopropyl, tert-butyl, isobutyl, 2-methylpropyl, cyclohexylmethyl, benzyl, thiazolyl-2-methyl, pyridyl-2-methyl, n-butyl, 2,2-dimethylpropyl, naphthylmethyl, or n-propyl group, or a substituted or unsubstituted phenyl group. Suitable phenyl substituents include one or more halogen, preferably fluorine, chlorine or bromine, atoms, C1-C4-alkyl groups, methoxy, ethoxy, nitro or trifluoromethyl groups or a dioxomethylene group. Alternately, R1g and R2g can, together with the xcex1-carbon atom, form a cyclopentane or cyclohexane ring or a benzo-fused cyclopentane ring, such as, for example, the indanyl group.
Identity of K
K is an xcex1-amino acid residue of Formula IIk, 
wherein R1k has the identity stated for R1k in Formula IIg, and R2k has the identity stated for R2g in Formula IIg,
Identity of L
In one embodiment, L is an amino or substituted amino group of Formula IIl, 
where R1l is a monovalent radical, such as a hydrogen atom, a normal or branched, saturated or unsaturated C1-C18-alkoxy group, a substituted or unsubstituted aryloxy group, a substituted or unsubstituted aryl-C1-C6-alkoxy group, or a substituted or unsubstituted aryloxy-C1-C6-alkoxy or heteroaryl-C1-C6-alkoxy group. The aryl group is preferably a phenyl or naphthyl group. The heteroaryl group is a 5-or 6-membered, preferably nitrogen-, oxygen-or sulfur-containing, ring system, such as, for example, a heteroaryl group derived from imidazole, isoxazole, isothiazole, thiazole, oxazole, pyrazole, thiophene, furan, pyrrole, 1,2,4-or 1,2,3-triazole, pyrazine, indole, benzofuran, benzothiophene, indole, isoindole, indazole, quinoline, pyridazine, pyrimidine, benzimidazole, benzopyran, benzothiazole, oxadiazole,thiadiazole or pyridine. Suitable aryl substituents include one or more halogen, preferably fluorine, bromine or chlorine, atoms, C1-C4-alkyl groups, methoxy, ethoxy or trifluoromethyl groups, a dioxymethylene group or nitro groups.
R2l is a monovalent radical, such as a hydrogen atom, a normal or branched, saturated or unsaturated C1-C18-alkyl group, a C3-C10-cycloalkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group. The aryl group is preferably a phenyl or naphthyl group. The heteroaryl group is a 5-or 6-membered, preferably nitrogen-, oxygen-or sulfur-containing, ring system, such as, for example, a heteroaryl group derived from imidazole, isoxazole, isothiazole, thiazole, oxazole, pyrazole, thiophene, furan, pyrrole, 1,2,4-or 1,2,3-triazole, pyrazine, indole, benzofuran, benzothiophene, indole, isoindole, indazole, quinoline, pyridazine, pyrimidine, benzimidazole, benzopyran, benzothiazole, oxadiazole,thiadiazole or pyridine. Suitable aryl substituents include one or more halogen, preferably fluorine, bromine or chlorine, atoms, C1-C4-alkyl groups, methoxy, ethoxy or trifluoromethyl groups, a dioxymethylene group or nitro groups.
R2l can, alternately, be of Formula IIl, 
where a, is an integer, such as 0, 1, 2, 3, 4 or 5. R3l is a monovalent radical, preferably a lower alkyl group, such as a methyl, ethyl, propyl or isopropyl group. R4l is a monovalent radical, which can be a saturated or partially unsaturated carbocyclic system comprising from about 3 to about 10 carbon atoms, a substituted or unsubstituted aryl or heteroaryl group, with aryl and heteroaryl and preferred substituents having the meaning stated for R2l in Formula IIl,
R2l can also be a substituent of Formula IIIr,
xe2x80x94(CH2)2xe2x80x94Wlxe2x80x94R5l xe2x80x83xe2x80x83(IIIr),
wherein Wl is an oxygen or sulfur atom or an N-R6l group. R5l is a monovalent radical, such as a hydrogen atom, a C1-C4-alkyl or C3-C7-cycloalkyl group or a substituted or unsubstituted aryl or arylmethyl group, with aryl and its preferred substituents having the meaning stated for R2l from Formula IIl. R6l is a monovalent radical, preferably a hydrogen atom, a C1-C4-alkyl group or a C3-C7-cycloalkyl group, a C1-C18-alkanoyl group, a benzoyl group or a substituted or unsubstituted aryl or arylmethyl group, with aryl and its preferred substituents having the meaning stated for R2l in Formula IIl.
R2l can, alternately, be a substituent of Formula IVr,
xe2x80x94(CH2)blxe2x80x94Zlxe2x80x83xe2x80x83(IVr),
where bl is an integer, preferably 2, 3 or 4. Zl can be a monovalent radical such as a formyl, aminocarbonyl or hydrazinocarbonyl group, or a cyclic or acyclic acetal or thioacetal group.
R2l can also be a substituent of Formula Vr, 
in which bl has the above-mentioned meaning. R7l can be a monovalent radical, such as a polyglycol group of the formula xe2x80x94Oxe2x80x94(CH2xe2x80x94CH2xe2x80x94O)dlxe2x80x94CH3, where dl is an integer, preferably in the range from about 2 to about 4 or from about 40 to about 90.
R2l can further be a carbohydrate of Formula VIr, 
where R8l is a monovalent radical, such as a hydrogen atom, a C1-C4-alkanoyl or alkyl group, a benzoyl group or a benzyl group.
L can also be a xcex2-hydroxylamino group of Formula IIIl, 
where R91 is a monovalent radical such as a hydrogen atom, a C1-C6-alkyl group or a substituted or unsubstituted aryl group, with aryl and its preferred substituents having the meaning stated for R2l . R10l is a monovalent radical, preferably a hydrogen atom, alkyl, for example, methyl, or a phenyl group.
When r and/or s is 1, L can also be an amino group of Formula IVl, 
where R2l and R4l are each a monovalent radical. R2l and R4l can also be linked by a carbon-carbon bond.
Another subclass of compounds of this invention includes peptides of Formula I wherein L is a hydrazido group of Formula Vl, 
and R11l is a monovalent radical, preferably a hydrogen atom. R12l can be a monovalent radical such as a hydrogen atom, a normal or branched C1-C8-alkyl group, a C3-C8-cycloalkyl group, a C3-C8-cycloalkyl-C1-C4-alkyl group or a substituted or unsubstituted aryl, heteroaryl, aryl-C1-C4-alkyl or heteroaryl-C1-C4-alkyl group, where aryl, heteroaryl and their preferred substituents can be selected from among the options listed for R2l.
When r and/or s is 1, R11l can also be selected from among the options listed above for R12l, and the two radicals together can additionally form a propylene or butylene bridge.
Another subclass of compounds of this invention includes peptides of Formula I wherein L is a monovalent radical of the formula xe2x80x94Oxe2x80x94R13l or the formula xe2x80x94Sxe2x80x94R13l, where R13l is a monovalent radical, such as a C3-C10-cycloalkyl group, a normal or branched C2-C6-alkenylmethyl group or a C1-C16-alkyl group which can be substituted by from 1 to about 5 halogen, preferably fluorine, atoms.
R13 can also be the radical xe2x80x94(CH2)exe2x80x94R14l, where e is an integer, preferably 1, 2 or 3. R14l is a monovalent radical, preferably a saturated or partially unsaturated C3-C10-carbocycle.
R13l can further be the monvalent radical xe2x80x94[CH2xe2x80x94CHxe2x95x90C(CH3)xe2x80x94CH2]lxe2x80x94H, where f is an integer, preferably 1, 2, 3 or 4.
R13l can also be the radical xe2x80x94[CH2xe2x80x94CH2xe2x80x94O]gxe2x80x94CH3, where g is an integer, preferably in the range from 1 to about 5.
R13l can also be the radical xe2x80x94(CH2)h-aryl or xe2x80x94(CH2)h-heteroaryl, where aryl and heteroaryl can also be substituted and, along with their preferred substituents, can be selected from the group listed for R2l. h is an integer, preferably 0, 1, 2 or 3.
R13l can further be the radical xe2x80x94(CH2)bxe2x80x94Wlxe2x80x94R5l. b, Wl and R5l can each be selected from among the options described for Formula IVl.
Another subclass of compounds of this invention includes peptides of Formula I in which L is an aminoxy group of the formula xe2x80x94Oxe2x80x94N(R5l)(R6l), where R15l and R6l are each a monovalent radical, which can independently be a hydrogen atom, a normal or branched C1-C8-alkyl group, which can be substituted by halogen, preferably fluorine, atoms, a C3-C8-cycloalkyl group, a C3-C8-cycloalkyl-C1-C4-alkyl group, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted aryl-C1-C4-alkyl group. Aryl and heteroaryl groups and the preferred substituents thereof can be selected from the options listed for R2l. R6, can be selected from among the options listed for R15l. Additionally, R15l and R16l can together form a 5-, 6-or 7-membered heterocycle. The compounds of the present invention further comprise the salts of the compounds described above with physiologically tolerated acids.
Another subclass of compounds of this invention includes peptides of Formula I wherein L is an oximato group of the formula xe2x80x94Oxe2x80x94Nxe2x95x90C(R15l)(R16l), R15l and R16l can be selected from among the options listed above and, additionally, can together form a cyclic system comprising, preferably, from about 3 to about 7 ring atoms. This cyclic system can additionally be fused to one or more aromatic rings. Particularly preferred cyclic systems are shown below. 
In one embodiment, the invention provides compounds of Formula I wherein A is an amino acid derivative selected from among N-alkyl-D-prolyl, N-alkyl-L-prolyl, N-alkyl-D-piperidine-2-carbonyl, N-alkyl-L-piperidine-2-carbonyl, N,N-dialkyl-D-2-ethyl-2-phenylglycyl and N,N-dialkyl-L-2-ethyl-2-phenylglycyl, wherein alkyl is methyl, ethyl or isopropyl; and B is a valyl, isoleucyl or 2-t-butyl-L-glycyl residue.
Preferred compounds of the invention include compounds of Formula I wherein r and s are each 0. A is an amino acid derivative selected from among D-N-methyl-piperidine-2-carbonyl, L-N-methyl-piperidine-2-carbonyl, N,N-dimethylamino-iso-butyryl, N-methyl-L-prolyl, N-methyl-L-thiazolidine-4-carbonyl, N,N-dimethyl-glycyl, L-prolyl, L-piperidine-2-carbonyl, N-propyl-D-piperidine-2-carbonyl, D-piperidine-2-carbonyl, N-ethyl-D-piperidine-2-carbonyl, N-methyl-[2,2,5,5-tetramethyl]-L-thiazolidine-2-carbonyl, N-isopropyl-D-piperidine-2-carbonyl, N,N-dimethyl-2-cyclopropylglycyl, N,N-dimethyl-L-2-ethyl-2-phenylglycyl, N,N-dimethyl-D-2-ethyl-2-phenylglycyl, D-prolyl, N-methyl-D-prolyl, N,N-dimethyl-2-(2-fluorophenyl)glycyl, 1-aza-[3,3,0]bicyclooctyl-5-carbonyl, N,N-dimethyl-2-[4-fluoro]phenyl-glycyl, N-methyl-[2,2,5,5-tetramethyl]-thiazolidine-2-carbonyl, 2-(R,S)-ethyl-2-phenylglycyl, D,L-1-aminoindane-1-carbonyl, N,N-dimethyl-2-(R,S)-methyl-2-phenylglycyl, 2-[N,N-dimethylamino]indane-2-carbonyl, 5-[N,N-dimethylamino]-5,6,7,8-tetrahydro-naphthalene-5-carbonyl, N-isopropyl-2-(R,S)-ethyl-2-phenylglycyl, 1-[N,N-dimethyl-amino]indane-2-carbonyl, N,N-dimethyl-2-propyl-2-phenylglycyl, N,N-dimethyl-2-[4-methoxy]phenyl-glycyl, N-methyl-3-hydroxy-D,L-valyl, N,N-dimethyl-D,L-2-isopropyl-2-phenylglycyl, N-methylpiperidine-2-carbonyl, N-methyl-L-prolyl, N-methyl-1,2,3,4-tetrahydroisoquinoline-1-carbonyl, N-methylazetidine-2-carbonyl, N-isopropylazetidine-2-carbonyl, N,N-dimethyl-[O-methyl]seryl, N,N-dimethyl-[O-methyl]threonyl, N-methyl-1,2,3,4-tetrahydroisoquinoline-3-carbonyl, 1-[N,N-dimethylamino]cyclohexyl-1-carbonyl, 1-[N,N-dimethylamino]cyclopentyl-1-carbonyl and 1,2,3,4-tetrahydroisoquinoline-3-carbonyl. B is valyl, isoleucyl or 2-tert-butylglycyl. D is N-methylvalyl, N-methyl-2-t-butylglycyl or N-methylisoleucyl. E and F are each, independently, prolyl, thiaprolyl, homoprolyl, hydroxyprolyl, 3,4-didehydroprolyl, 4-fluoroprolyl, and 3-methylprolyl. L is an alkoxy group or an amino group of the formula R1l-N-R2L, wherein R1l and R2l are independently selected from the group consisting of hydrogen, alkoxy, hydroxy, alkyl and alkylaryl.
In a particularly preferred subset of the compounds of the invention, r and s are each 0. A is an amino acid derivative selected from among D-N-methyl-piperidine-2-carbonyl, N-ethyl-D-piperidine-2-carbonyl, N-isopropyl-D-piperidine-2-carbonyl, N,N-dimethyl-2-cyclopropyl-glycyl, N-methyl-D-prolyl, 1-aza-[3,3,0]bicyclooctyl-5-carbonyl, N-methyl-[2,2,5,5-tetramethyl]-thiazolidine-2-carbonyl, 2-(R,S)-ethyl-2-phenylglycyl, D,L-1-aminoindane-1-carbonyl, N,N-dimethyl-2-(R,S)-methyl-2-phenylglycyl, 5-[N,N-dimethylamino]-5,6,7,8-tetrahydro-naphthalene-5-carbonyl, 1-[N,N-dimethylamino]indane-2-carbonyl, N,N-dimethyl-2-propyl-2-phenylglycyl, N,N-dimethyl-L-2-ethyl-2-phenylglycyl, N,N-dimethyl-D-2-ethyl-2-phenylglycyl, N-methyl-3-hydroxy-D,L-valyl, N,N-dimethyl-D,L-2-isopropyl-2-phenylglycyl, N-methyl-piperidine-2-carbonyl, N-methyl-D,L-prolyl, N-methyl-1,2,3,4-tetra-hydroisoquinoline-1-carbonyl, N-methylazetidine-2-carbonyl, N-isopropylazetidine-2-carbonyl, N,N-dimethyl-[O-methyl]seryl, 1-[N,N-dimethylamino]cyclohexyl-1-carbonyl and 1-[N,N-dimethylamino]cyclopentyl-1-carbonyl. B is valyl; D is N-methylvalyl; and E and F are each prolyl. L is a C1-C6-alkoxy group or an amino group of the formula R1l xe2x80x94Nxe2x80x94R21, wherein R1l and R2l are each independently selected from the group consisting of hydrogen, C1-C6-alkoxy, hydroxy, normal, cyclic or branched C1-C12-alkyl, and phenylalkyl.
The compounds of the present invention can be prepared by known methods of peptide synthesis. Thus, the peptides can be assembled sequentially from individual amino acids or by linking suitable small peptide fragments. In sequential assembly, the peptide chain is extended stepwise, starting at the C-terminus, by one amino acid per step. In fragment coupling, fragments of different lengths can be linked together, and the fragments in turn can be obtained by sequential assembly from amino acids or by fragment coupling of still shorter peptides.
In both sequential assembly and fragment coupling it is necessary to link the units by forming an amide linkage, which can be accomplished via a variety of enzymatic and chemical methods. Chemical methods for forming the amide linkage are described in detail in standard references on peptide chemistry, including Mxc3xciller, Methoden der organischen Chemie Vol. XV/2, 1-364, Thieme Verlag, Stuttgart, (1974); Stewart and Young, Solid Phase Peptide Synthesis, 31-34 and 71-82, Pierce Chemical Company, Rockford, Ill. (1984); Bodanszky et al., Peptide Synthesis, 85-128, John Wiley and Sons, New York, (1976). Preferred methods include the azide method, the symmetric and mixed anhydride method, the use of in situ generated or preformed active esters, the use of urethane protected N-carboxy anhydrides of amino acids and the formation of the amide linkage using coupling reagents, such as carboxylic acid activators, especially dicyclohexylcarbodiimide (DCC), diusopropylcarbodiimide (DIC), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), pivaloyl chloride, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl), n-propanephosphonic anhydride (PPA), N,N-bis(2-oxo-oxazolidinyl)amidophosphoryl chloride (BOP-Cl), bromo-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBrop), diphenyl-phosphoryl azide (DPPA), Castro""s reagent (BOP, PyBop), O-benzotriazolyl-N,N,Nxe2x80x2, Nxe2x80x2-tetramethyluronium salts (HBTU), O-azabenzotriazolyl-N,N,Nxe2x80x2,Nxe2x80x2-tetramethyluronium salts (HATU), diethylphosphoryl cyanide (DEPCN), 2,5-diphenyl-2,3-dihydro-3-oxo-4-hydroxythiophene dioxide (Steglichxe2x80x2s reagent; HOTDO), and 1,1xe2x80x2-carbonyldi-imidazole (CDI). The coupling reagents can be employed alone or in combination with additives such as N,N-dimethyl-4-aminopyridine (DMAP), N-hydroxy-benzotriazole (HOBt), N-hydroxyazabenzotriazole (HOAt), N-hydroxybenzotriazine (HOOBt), N-hydroxysuccinimide (HOSu) or 2-hydroxypyridine.
Although the use of protecting groups is generally not necessary in enzymatic peptide synthesis, reversible protection of reactive groups not involved in formation of the amide linkage is necessary for both reactants in chemical synthesis. Three conventional protective group techniques are preferred for chemical peptide synthesis: the benzyloxycarbonyl (Z), the t-butoxycarbonyl (Boc) and the 9-fluorenylmethoxy-carbonyl (Fmoc) techniques. Identified in each case is the protective group on the xcex1-amino group of the chain-extending unit. A detailed review of amino-acid protective groups is given by Mxc3xcller, Methoden der orjanischen Chemie Vol. XV/1, pp 20-906, Thieme Verlag, Stuttgart (1974). The units employed for assembling the peptide chain can be reacted in solution, in suspension or by a method similar to that described by Merrifield, J. Am. Chem. Soc. 85: (1963) 2149.
Solvents suitable for peptide synthesis include any solvent which is inert under the reaction conditions, especially water, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile, dichloromethane (DCM), 1,4-dioxane, tetrahydrofuran (THF), N-methyl-2-pyrrolidone (NMP) and mixtures of these solvents.
Peptide synthesis on the polymeric support can be carried out in a suitable inert organic solvent in which the amino acid derivatives starting materials are soluble. However, preferred solvents additionally have resin-swelling properties, such as DMF, DCM, NMP, acetonitrile and DMS0, and mixtures of these solvents. Following synthesis, the peptide is removed from the polymeric support. The conditions under which this cleavage is accomplished for various resin types are disclosed in the literature. The cleavage reactions most commonly used areacid-or palladium-catalyzed, the former being conducted in, for example, liquid anhydrous hydrogen fluoride, anhydrous trifluoromethanesulfonic acid, dilute or concentrated trifluoroacetic acid, and acetic acid/dichloromethane/trifluoroethanol mixtures. The latter can be carried out in THF or THF-DCM-mixtures in the presence of a weak base, such as morpholine. Certain protecting groups are also cleaved off under these conditions.
Partial deprotection of the peptide may also be necessary prior to certain derivatization reactions. For example, peptides dialkylated at the N-terminus can be prepared by coupling the appropriate N,N-di-alkylamino acid to the peptide in solution or on the polymeric support, by reductive alkylation of the resin-bound peptide in DMF/1% acetic acid with NaCNBH3 and the appropriate aldehyde or by hydrogenation of the peptide in solution in the presence of the appropriate aldehyde or ketone and Pd/carbon.
The various non-naturally occurring amino acids as well as the various non-amino acid moieties disclosed herein can be obtained from commercial sources or synthesized from commercially available staring materials using methods known in the art. For example, amino acid building blocks with R1 and R2 groups can be prepared according to the method described by Wuensch and Weyl, Methoden der Organische Chemie, vol. XV, Springer Verlag: Stuttgart, p. 306 (1974) and references cited therein.
In another embodiment, the present invention comprises a method for partially or totally inhibiting formation of, or otherwise treating (e.g., reversing or inhibiting the further development of) solid tumors (e.g., tumors of the lung, breast, colon, prostate, bladder, rectum, or endometrial tumors) or hematological malignancies (e.g., leukemias, lymphomas) in a mammal, for example, a human, by administering to the mammal a therapeutically effective amount of a compound or a combination of compounds of Formula I. The compound(s) may be administered alone or in a pharmaceutical composition comprising the compound(s) and an acceptable carrier or diluent. Administration can be by any of the means which are conventional for pharmaceutical, preferably oncological, agents, including oral and parenteral means, such as subcutaneously, intravenously, intramuscularly and intraperitoneally, nasally or rectally. The compounds may be administered alone or in the form of pharmaceutical compositions containing a compound or compounds of Formula I together with a pharmaceutically accepted carrier appropriate for the desired route of administration. Such pharmaceutical compositions may be combination products, i.e., they may also contain other therapeutically active ingredients.
The dosage to be administered to the mammal, such as a human, will contain a therapeutically effective amount of a compound described herein. As used herein, xe2x80x9ctherapeutically effective amountxe2x80x9d is an amount sufficient to inhibit (partially or totally) formation of a tumor or a hematological malignancy or to reverse development of a solid tumor or other malignancy or prevent or reduce its further progression. For a particular condition or method of treatment, the dosage is determined empirically, using known methods, and will depend upon factors such as the biological activity of the particular compound employed; the means of administration; the age, health and body weight of the recipient; the nature and extent of the symptoms; the frequency of treatment; the administration of other therapies; and the effect desired. A typical daily dose will be from about 0.05 to about 50 milligrams per kilogram of body weight by oral administration and from about 0.01 to about 20 milligrams per kilogram of body weight by parenteral administration.
The compounds of the present invention can be administered in conventional solid or liquid pharmaceutical administration forms, for example, uncoated or (film-)coated tablets, capsules, powders, granules, suppositories or solutions. These are produced in a conventional manner. The active substances can for this purpose be processed with conventional pharmaceutical aids such as tablet binders, fillers, preservatives, tablet disintegrants, flow regulators, plasticizers, wetting agents, dispersants, emulsifiers, solvents, sustained release compositions, antioxidants and/or propellant gases (cf. H. Sxc3xccker et al.: Pharmazeutische Technologie, Thieme-Verlag, Stuttgart, 1978). The administration forms obtained in this way typically contain from about 1 to about 90% by weight of the active substance.
The present invention will now be illustrated by the following examples, which are not limiting.